Method for detecting trifluridine-related substance by high-performance liquid chromatography

ABSTRACT

The present invention provides a novel method that is capable of detecting a trifluridine-related substance from a sample containing trifluridine or a salt thereof by high-performance liquid chromatography comprising two steps that are performed under gradient conditions. More specifically, the method is for detecting a trifluridine-related substance, the method comprising the step of subjecting a sample containing trifluridine or a salt thereof to high-performance liquid chromatography using a mobile phase composed of an organic phase and an aqueous phase, wherein the step of high-performance liquid chromatography comprises steps 1 and 2 that satisfy the following requirements: Step 1: the percentage of the organic phase in the entire mobile phase is 1 to 14% by volume; and Step 2: after step 1, elution is performed by applying a gradient of increasing the percentage of the organic phase in the entire mobile phase.

TECHNICAL FIELD

The present invention relates to a method for measuring atrifluridine-related substance contained in a pharmaceutical preparationcontaining trifluridine by high-performance liquid chromatography.

BACKGROUND ART

Related substances in medicinal drugs are generally measured by liquidchromatography, and a normal- or reversed-phase column system iscommonly used with a mobile phase composed of a liquid mixture of waterand an organic solvent. Trifluridine has long been used as an ophthalmicdrop for infection treatment under the name of Viropic (registeredtrademark), and Lonsurf (registered trademark), a product of theApplicant, is a pharmaceutical preparation comprising trifluridine andtipiracil. The medicinal drug comprising trifluridine and tipiracil issold as an antitumor agent, and there have been reports on thispharmaceutical preparation (Patent Literature (PTL) 1, PTL 2, PTL 3, andPTL 4).

Known trifluridine-related substances are 5-trifluoromethyluracil and5-carboxyuracil, and Non-Patent Literature (NPL) 1 reports the use ofhigh-performance liquid chromatography to measure these relatedsubstances.

NPL 2 reports that 5-trifluoromethyluracil, which is atrifluridine-related substance, can be measured by reversed-phase liquidchromatography under various conditions.

To measure the amount of trifluridine incorporated into tumor cells, NPL3 reports using liquid chromatography.

To determine the substrate specificity of TK1 of trifluridine, NPL 4reports using liquid chromatography.

To measure trifluridine in enzyme synthesis, NPL 5 reports using liquidchromatography.

Further, NPL 6, NPL 7, and NPL 8 report quantitative analysis ofmedicinal drugs that contain trifluridine and tipiracil by liquidchromatography.

To measure 5-trifluoromethyluracil, which is a trifluridine-relatedsubstance, NPL 2 reports various conditions for high-performance liquidchromatography using acetonitrile.

To measure the amount of trifluridine incorporated into tumor cells, NPL3 reports conditions for high-performance liquid chromatography usingacetonitrile.

To determine the substrate specificity of TK1 of trifluridine, NPL 4reports conditions for high-performance liquid chromatography usingacetonitrile.

To confirm trifluridine in enzyme synthesis, NPL 5 reports conditionsfor high-performance liquid chromatography with the use of trimethylammonium acetate in a gradient mode.

For quantitative measurement of trifluridine and tipiracil contained inLonsurf, NPL 6, NPL 7, and NPL 8 report conditions for high-performanceliquid chromatography using acetonitrile.

Regarding trifluridine as eye drops, NPL 9 and NPL 10 report conditionsfor high-performance liquid chromatography using methanol.

Regarding trifluridine, NPL 11, NPL 12, PTL 6, and PTL 13 reportconditions for high-performance liquid chromatography using a mobilephase to which an acetate buffer, trifluoroacetic acid, or acetic acidis added.

Further, PTL 7 discloses performing high-performance liquidchromatography to confirm the purity of trifluridine; however, PTL 7nowhere discloses conditions for the chromatography.

However, these reports nowhere suggest a method for detecting atrifluridine-related substance by high-performance liquid chromatographycomprising two steps that are performed under gradient conditions.

CITATION LIST Patent Literature

-   PTL 1: WO2013/122134-   PTL 2: WO2013/122135-   PTL 3: WO2006/080327-   PTL 4: WO96/30346-   PTL 5: CN106749194A-   PTL 6: CN105198947A-   PTL 7: CN105461772A-   PTL 8: Japanese Patent No. 4603274-   PTL 9: Japanese Patent No. 4441313-   PTL 10: Japanese Patent No. 4437786

Non-Patent Literature

-   NPL 1: P. Horsch et al., International Journal of Pharmaceutics 222    (2001), pp. 205-215-   NPL 2: D. V. Moiseev et al., Pharmaceutical Chemistry Journal 41, 1    (2007), pp. 25-33-   NPL 3: N. TANAKA et al., Oncology Reports 32 (2014), pp. 2319-2326-   NPL 4: K. SAKAMOTO et al., International Journal of Oncology 46    (2015), pp. 2327-2334-   NPL 5: A. Fresco-Taboada et al., Catalysis Today 259 (2015), pp.    197-204-   NPL 6: M. S. H. Rizwan et al., International Journal of Innovative    Pharmaceutical Sciences and Research 5 (2017), pp. 32-42-   NPL 7: S. GODAY et al., International Journal of Research in    Applied, Natural and Social Sciences 5 (2017), pp. 93-104-   NPL 8: K. Jogi et al., International Journal of Research in Pharmacy    and Chemistry 7 (2017), pp. 63-70-   NPL 9: B. Paw et al., Pharmazie 7 (1997), pp. 551-552-   NPL 10: T. Briggle et al., Journal of Chromatography 381 (1986), pp.    343-355-   NPL 11: M. Riegel et al., Journal of Chromatography 568 (1991), pp.    467-474-   NPL 12: G. Balansard et al., Journal of Chromatography 348 (1985),    pp. 299-303-   NPL 13: T. Kawauchi et al., Journal of Chromatography 751 (2001),    pp. 325-330

SUMMARY OF INVENTION Technical Problem

The problem to be solved by the present invention is to provide a novelmethod that is capable of detecting a trifluridine-related substancefrom a sample containing trifluridine or a salt thereof byhigh-performance liquid chromatography comprising two steps that areperformed under gradient conditions.

Solution to Problem

The present inventors conducted extensive research and found a methodthat is capable of efficiently detecting related substances oftrifluridine or a salt thereof by high-performance liquid chromatographyunder specific conditions, and found that the method is suitable forassuring the quality thereof.

Therefore, the present invention provides the following Items 1 to 16.

1. A method for detecting a trifluridine-related substance, the methodcomprising the step of subjecting a sample containing trifluridine or asalt thereof to high-performance liquid chromatography using a mobilephase composed of an organic phase and an aqueous phase, wherein thestep of high-performance liquid chromatography comprises steps 1 and 2that satisfy the following requirements:

Step 1: the percentage of the organic phase in the entire mobile phaseis 1 to 14% by volume; and

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase in the entire mobilephase.

2. The method according to Item 1, wherein the related substance is atleast one member selected from the group consisting of the followingrelated substances 1 to 6:

related substance 1: 5-carboxyuracil,

related substance 2: 5-carboxy-2′-deoxy-uridine,

related substance 3: 2′-deoxy-5-methoxycarbonyluridine,

related substance 4: trifluorothymine,

related substance 5: 5-methoxycarbonyluracil, and

related substance 6:5′-(4-chlorophenylcarboxy)-2′-deoxy-5-trifluoromethyluridine.

3. The method according to Item 1 or 2, wherein related substances 1 to5 are detected in step 1, and related substance 6 is detected in step 2.

4. The method according to any one of Items 1 to 3, wherein step 1 isperformed under isocratic conditions.

5. The method according to any one of Items 1 to 4, wherein the organicphase is acetonitrile.

6. The method according to any one of Items 1 to 5, wherein, in step 1,the percentage of the organic phase in the entire mobile phase is withina range of 2 to 10% by volume.

7. The method according to any one of Items 1 to 6, wherein thepercentage of the organic phase in the entire mobile phase at the end ofstep 2 is within a range of 25 to 70% by volume.

8. The method according to any one of Items 1 to 7, wherein, in step 2,the elution is performed by applying a gradient of increasing thepercentage of the organic phase in the entire mobile phase by 0.9% byvolume or more per 1 minute.

9. The method according to any one of Items 1 to 8, wherein themeasurement time in step 2 is within a range of 10 to 50 minutes.

10. The method according to any one of Items 1 to 9, wherein the flowrate at the end of step 2 is 1.0 to 1.5 times the flow rate in step 1.

11. The method according to any one of Items 1 to 10, wherein theaqueous phase further contains phosphate.

12. The method according to any one of Items 1 to 11, wherein theaqueous phase further contains methanol.

13. 5-Carboxy-2′-deoxy-uridine (related substance 2) for use in qualitycontrol of a combination drug containing trifluridine or a salt thereof.

14. 5-Carboxy-2′-deoxy-uridine (related substance 2) for use as astandard for detecting impurities in a combination drug containingtrifluridine or a salt thereof.

15. 2′-Deoxy-5-methoxycarbonyluridine (related substance 3) for use inquality control of a combination drug containing trifluridine or a saltthereof.

16. 2′-Deoxy-5-methoxycarbonyluridine (related substance 3) for use as astandard for detecting impurities in a combination drug containingtrifluridine or a salt thereof.

17. 5-Methoxycarbonyluracil (related substance 5) for use in qualitycontrol of a combination drug containing trifluridine or a salt thereof.

18. 5-Methoxycarbonyluracil (related substance 5) for use as a standardfor detecting impurities in a combination drug containing trifluridineor a salt thereof.

19. 5′-(4-Chlorophenylcarboxy)-2′-deoxy-5-trifluoromethyluridine(related substance 6) for use in quality control of a combination drugcontaining trifluridine or a salt thereof.

20. 5′-(4-Chlorophenylcarboxy-2′-deoxy-5-trifluoromethyluridine (relatedsubstance 6) for use as a standard for detecting impurities in acombination drug containing trifluridine or a salt thereof.

Advantageous Effects of Invention

According to the method of the present invention, related substances oftrifluridine or a salt thereof can be efficiently detected byhigh-performance liquid chromatography using a mobile phase composed ofan organic phase and an aqueous phase. Further, the method of thepresent invention is suitable for assuring the quality thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the chromatogram obtained in Example 1-1.

FIG. 2 shows the chromatogram obtained in Example 1-3.

FIG. 3 shows the chromatogram obtained in Example 2-1.

DESCRIPTION OF EMBODIMENTS

Trifluridine (FTD) referred to in the present invention isα,α,α,-trifluorothymidine and is a compound having the followingstructure:

In the present invention, when trifluridine has isomers, such as opticalisomers, stereoisomers, rotational isomers, and tautomers, all of theisomers and mixtures thereof are encompassed within the scope of thecompound of the present invention, unless otherwise stated.

In the present invention, a salt refers to a pharmaceutically acceptablesalt, unless otherwise stated, and may be a base addition salt or anacid addition salt.

Examples of base addition salts include alkali metal salts, such assodium salts and potassium salts; alkaline earth metal salts, such ascalcium salts and magnesium salts; ammonium salts; and organic aminesalts, such as trimethylamine salts, triethylamine salts,dicyclohexylamine salts, ethanolamine salts, diethanolamine salts,triethanolamine salts, procaine salts, and N,N′-dibenzylethylenediaminesalts.

Examples of acid addition salts include inorganic acid salts, such ashydrochloride, sulfate, nitrate, phosphate, and perchlorate; organicacid salts, such as acetate, formate, maleate, fumarate, tartrate,citrate, ascorbate, and trifluoroacetate; and sulfonates, such asmethanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate.

The trifluridine or a salt thereof used in the present invention ispreferably trifluridine in the free, non-salt foLnL.

The method of the present invention is capable of detecting atrifluridine-related substance by preparing a sample containing asolvent and trifluridine or a salt thereof, and subjecting the sample tohigh-performance liquid chromatography.

The sample according to the present invention can contain atrifluridine-related substance. Examples of the related substancesinclude the compounds shown as related substances 1 to 6.

TABLE 1 Related Substance Compound Name Structural Formula 15-Carboxyuracil

2 5-Carboxy-2′-deoxy- uridine

3 2′-Deoxy-5- methoxycarbonyluridine

4 Trifluorothymine

5 5 -Methoxycarbonyluracil

6 5′-(4- Chlorophenylcarboxy-2′- deoxy-5- trifluoromethyluridine)

Related substance 1 is 5-carboxyuracil. Related substance 1 may besometimes referred to as2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid.

Related substance 2 is 5-carboxy-2′-deoxy-uridine. Related substance 2may be sometimes referred to as1-((2R,4R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylicacid.

Related substance 3 is 2′-deoxy-5-methoxycarbonyluridine. Relatedsubstance 3 may be sometimes referred to as1-((2R,4R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-methylcarboxylic acid.

Related substance 4 is trifluorothymine. Related substance 4 may besometimes referred to as 5-(trifluoromethyl)pyrimidine-2,4(1H,3H)-dione.

Related substance 5 is 5-methoxycarbonyluracil. Related substance 5 maybe sometimes referred to as2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-methyl carboxylic acid.

Related substance 6 is5′-(4-chlorophenylcarboxy-2′-deoxy-5-trifluoromethyluridine). Relatedsubstance 6 may be sometimes referred to as((2R,3R,5R)-5-(2,4-dioxo-5-(trifluoromethyl)-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl4-chlorobenzoate.

In addition to samples prepared from a pharmaceutical preparation ordrug substance itself, it is possible in the present invention to usesamples prepared in a test for determining stability etc., samplesprepared by adding each related substance to study the retention timeetc. of the related substance, samples prepared to confirm themanufacturing process of a pharmaceutical preparation or drug substance,and the like. The sample may contain or may not substantially containtipiracil or a salt thereof contained in Lonsurf (registered trademark).

The pharmaceutical preparation usable in the present invention containstrifluridine or a salt thereof and optionally a pharmaceuticallyacceptable carrier, and may be formed into a suitable dosage formaccording to prevention and treatment purposes. Examples of the dosageform include oral preparations, ophthalmic solutions, injections,suppositories, ointments, patches, and the like, with oral preparationsbeing preferable. Such dosage forms can be formed by methodsconventionally known to persons skilled in the art.

As the pharmaceutically acceptable carrier, various conventional organicor inorganic carrier materials used as preparation materials may beincorporated as an excipient, binder, disintegrant, lubricant, orcolorant in solid preparations; or as a solvent, solubilizing agent,suspending agent, isotonizing agent, buffer, or soothing agent in liquidpreparations. It is also possible to use pharmaceutical preparationadditives, such as antiseptics, antioxidants, colorants, sweeteners, andstabilizers, if required.

Oral solid preparations when prepared are produced by adding anexcipient and optionally a binder, disintegrant, lubricant, colorant,taste-masking or flavoring agent, etc., to trifluridine or a saltthereof, and formulating the resulting mixture into tablets, coatedtablets, granules, powders, capsules, or the like by ordinary methods.

Ophthalmic solutions when prepared are produced by using trifluridine ora salt thereof with a solubilizing agent, an isotonizing agent, abuffer, an antiseptic, and the like.

The daily dose of the medicinal drug in such a dosage form may besuitably determined according to the condition, body weight, age,gender, etc., of the patient.

To perform high-performance liquid chromatography in the presentinvention, a commercially available chromatography device can be used.

Known columns for chromatography include normal-phase columns, in whichan organic phase is used as the mobile phase to separate lipophiliccompounds, and reversed-phase columns, in which an aqueous phase is usedas the mobile phase to separate compounds. In high-performance liquidchromatography, a reversed-phase column is often used. In the presentinvention as well, reversed-phase chromatography is preferable.

The column for high-performance liquid chromatography usable in thepresent invention is selected from a silica gel column, a columncontaining silica gel whose surface is modified with octadecyl silylgroups (an ODS column or C18 column), a column containing silica gelwhose surface is modified with octyl groups (a C8 column), a columncontaining silica gel whose surface is modified with cyanopropyl groups(a CN column), a column containing silica gel whose surface is modifiedwith phenethyl groups (a Ph column), a column containing silica gelwhose surface is modified with aminopropyl groups (an NH column), acolumn containing silica gel whose surface is modified withdihydroxypropyl groups (a Diol column), a column packed with variouspolymers (a polymer column), a column packed with ion-exchange resin (anion-exchange column), and the like. In the present invention, an ODScolumn is preferable.

It is possible to use various types of ODS columns with different silicagel particle sizes, different pore sizes, different types of bonding ofoctadecyl silyl groups, different degrees of substitution of octadecylsilyl groups, etc. In the present invention, a high-purity silica gel isused, and it is preferable to use an ODS column (an end-capped ODScolumn) in which residual silanol obtained after octadecylation istreated with a low-molecular-weight silylating agent.

It is possible to use various types of ODS columns with different silicagel particle sizes, different pore sizes, different types of bonding ofoctadecyl silyl groups, different degrees of substitution of octadecylsilyl groups, etc. In the present invention, the silica gel preferablyhas an average particle size of, for example, 2 to 10 μm, and morepreferably 3 to 5 μm. The average particle size of silica gel can bemeasured by, for example, laser diffractometry. The silica gel has anaverage pore size of, for example, 6 to 20 nm, and more preferably 8 to13 nm. The average pore size of silica gel can be measured by a gasabsorption method etc. The bonding type of octadecyl silyl groups in thesilica gel is preferably, for example, monomeric or polymeric. Thedegree of substitution of octadecyl silyl groups can be measured byvarious methods. The carbon content in the silica gel is preferably, forexample, 3% or more, and more preferably 10% or more. The carbon contentin the silica gel is preferably, for example, 25% or less, and morepreferably 20% or less. The carbon content in the silica gel can bemeasured by various methods.

Examples of the organic phase used in the mobile phase inhigh-performance liquid chromatography include non-polar solvents, suchas hexane, cyclohexane, heptane, diethyl ether, tetrahydrofuran,chloroform, and methylene chloride; aprotic polar solvents, such asacetone, dimethylsulfoxide, and acetonitrile; acetic acid; methanol;ethanol; isopropanol; acetonitrile; and the like. These solvents may beused alone, or two or more of these solvents may be used as a mixedsolvent. The organic phase according to the present invention ispreferably methanol or acetonitrile, and more preferably acetonitrile.The organic phase may contain 10% or less of water.

The aqueous phase used in the mobile phase in high-performance liquidchromatography may contain 10% or less of an organic solvent in additionto water. The aqueous phase preferably contains 10% or less of methanol,more preferably 5% or less of methanol, still more preferably 2% or lessof methanol, and particularly preferably 0.1 to 1% of methanol, in theentire aqueous phase.

To enable reproducibility, various buffers can be added to the mobilephase in high-performance liquid chromatography. For example, it ispossible to add acetic acid or a salt thereof, citric acid or a saltthereof, tartaric acid or a salt thereof, and phosphoric acid or a saltthereof. Examples of acetic acid or a salt thereof include acetic acidand sodium acetate. Examples of citric acid or a salt thereof includecitric acid, monosodium citrate, disodium citrate, and trisodiumcitrate. Examples of tartaric acid or a salt thereof include tartaricacid and sodium tartrate. Examples of phosphoric acid or a salt thereofinclude phosphoric acid, sodium dihydrogenphosphate, disodiumhydrogenphosphate, potassium dihydrogen phosphate, and dipotassiumhydrogen phosphate. Additives in the aqueous phase according to thepresent invention are preferably phosphate, and more preferably sodiumdihydrogenphosphate, from the viewpoint of the properties of thesubstances to be measured, the shape of the peaks obtained by themeasurement, as well as the measurement reproducibility. These additivesmay be used alone or in a combination of two or more.

The concentration of the buffer that can be used in the presentinvention may be suitably adjusted within a concentration range in whichthe buffer does not undergo precipitation during the high-performanceliquid chromatography measurement. The concentration is preferably 1 to50 mM, more preferably 5 to 40 mM, still more preferably 10 to 30 mM,and particularly preferably 18 to 20 mM.

For high-performance liquid chromatography, a mixture of an organicphase and an aqueous phase is used as the mobile phase. In the presentinvention, the percentage of the organic phase in the entire mobilephase is preferably within a range of 1 to 14% by volume, morepreferably 2 to 10% by volume, and particularly preferably 3 to 7% byvolume at the retention times of related substances 1, 2, 3, 4, and 5,and trifluridine.

As the mobile phase in high-performance liquid chromatography, a mixtureof an organic phase and an aqueous phase is used. The ratio thereof isoften made to vary during the measurement, and this is referred to as agradient application.

The gradient application is usually performed often in consideration ofthe retention time of the target compound and the separation of thetarget compound and related substances from each other.

The present invention is characterized by gradient conditions of steps 1and 2.

Step 1: The percentage of the organic phase in the entire mobile phaseis 1 to 14% by volume.

Step 2: After step 1, elution is performed by applying a gradient offurther increasing the percentage of the organic phase in the entiremobile phase.

In step 1, the mobile phase may be used in a gradient or isocratic mode,and is preferably used in an isocratic mode. Further, in step 1, thepercentage of the organic phase in the entire mobile phase may be, forexample, 1 to 14% by volume, more preferably 2 to 10% by volume, andparticularly preferably 3 to 7% by volume. In the present invention, thephrase “in step 1, the percentage of the organic phase in the entiremobile phase is X to Y % by volume” means that the percentage thereof iswithin a range of X to Y % by volume during step 1.

Step 1 is initiated within 5 minutes of, preferably within 3 minutes of,more preferably within 1 minute of, and particularly preferablysimultaneously with, the injection of the sample into thehigh-performance liquid chromatography.

Step 1 is completed 13 to 30 minutes after the sample is injected intothe high-performance liquid chromatography. Step 1 is preferablycompleted 15 to 28 minutes and preferably 17 to 25 minutes after theinjection, so that trifluridine does not overlap with valleys or ghostpeaks in the baseline.

The measurement time in step 1 is not particularly limited as long as itis within the range between the initiation and completion stated above.The measurement time is preferably 15 to 28 minutes, and more preferably17 to 25 minutes.

The upper limit of the flow rate of the mobile phase in step 1 is notlimited as long as it is commonly applied to high-performance liquidchromatography. In consideration of the separation of the retention timeof each related substance, the flow rate is preferably 2.5 mL/min orless, more preferably 2.0 mL/min or less, still more preferably 1.5mL/min or less, yet more preferably 1.3 mL/min or less, and particularlypreferably 1.1 mL/min or less. The range of the flow rate of the mobilephase in step 1 is not particularly limited. For example, it ispreferably 0.5 to 2.5 mL/min, more preferably 0.5 to 2.0 mL/min, stillmore preferably 0.7 to 1.5 mL/min, yet more preferably 0.8 to 1.3mL/min, and particularly preferably 0.9 to 1.1 mL/min.

In this manner, in step 1, at least one trifluridine-related substanceselected from the group consisting of related substances 1, 2, 3, 4, and5 can be detected. In step 1, it is preferable that related substances1, 2, 3, 4, and 5 be detected.

From the viewpoint of performing step 1 under more isocratic conditions,when at least one member from among related substances 1, 2, 3, 4, and 5is detected in step 1, the difference between the maximum value and theminimum value of the percentage of the organic phase in the entiremobile phase is preferably 5% by volume or less, and particularlypreferably 1% by volume or less, with respect to the entire mobile phaseat the retention time of each substance detected in step 1 from amongrelated substances 1, 2, 3, 4, and 5.

In step 2, the percentage of the organic phase is increased with amobile-phase gradient. If step 1 is performed under gradient conditions,then a gradient is applied in step 2 so as to further increase thepercentage of the organic phase. If step 1 is performed under isocraticconditions, then a gradient is applied in step 2 to increase thepercentage of the organic phase.

The percentage of the organic phase in the entire mobile phase at theend of step 2 is 25 to 70% by volume, more preferably 30 to 65% byvolume, and particularly preferably 35 to 60% by volume. In the presentinvention, the phrase “at the end of step 2” refers to the time at whichthe gradient application is stopped so that the percentage of theorganic phase in the entire mobile phase starts to decrease in step 2.

When to start step 2 is not particularly limited as long as step 2 isstarted within 3 minutes after the completion of step 1. Step 2 ispreferably started within 1 minute after, and preferably simultaneouslywith, the completion of step 1.

The completion of step 2 is not particularly limited as long as it isafter the retention time of related substance 6. Step 2 is preferablycompleted 35 to 65 minutes, and preferably 40 to 60 minutes after theinjection of the sample into the high-performance liquid chromatography.

The measurement time in step 2 is not particularly limited as long asthe retention time of related substance 6 can be measured, and ispreferably 10 to 50 minutes, and more preferably 15 to 45 minutes.

The gradient in step 2 is not particularly limited as long as thepercentage of the organic phase in the entire mobile phase is increased.The percentage of the organic phase in the entire mobile phase ispreferably increased by 0.9% by volume or more per 1 minute, morepreferably increased by 1.0% by volume or more per 1 minute, andparticularly preferably increased by 2.0% by volume or more per 1minute. The upper limit of the increase in the percentage of the organicphase in the entire mobile phase is not particularly limited, and ispreferably, for example, 10% by volume or less per 1 minute, and morepreferably, for example, 5.0% by volume or less per 1 minute. In thisspecification, when the percentage of the organic phase in the entiremobile phase is increased by X % by volume per unit time (e.g., per 1minute), the percentage of the organic phase in the entire mobile phaseis increased by X % by volume with respect to 100% by volume of theentire mobile phase per unit time. Further, in the present invention,the change in the percentage of the organic phase in the entire mobilephase during the measurement in step 2 (the difference between themaximum value and the minimum value of the percentage of the organicphase in the entire mobile phase during the measurement) is preferably30% by volume or more, and particularly preferably 50% by volume ormore, with respect to the entire mobile phase. The upper limit of thechange in the percentage of the organic phase in the entire mobile phaseduring the measurement in step 2 is not particularly limited, and ispreferably, for example, 80% by volume or less, and particularlypreferably 70% by volume or less, with respect to the entire mobilephase.

The flow rate of the mobile phase in step 2 is not limited as long as itis commonly applied to high-performance liquid chromatography. Inconsideration of the separation of the retention time of each relatedsubstance, the flow rate is preferably 2.5 mL/min or less, morepreferably 2.0 mL/min or less, and still more preferably 1.5 mL/min orless. The range of the flow rate of the mobile phase in step 2 is notparticularly limited, and is, for example, preferably 0.5 to 2.5 mL/min,more preferably 0.5 to 2.0 mL/min, and still more preferably 1.0 to 1.5mL/min. To shorten the entire measurement time, it is possible togradually increase the flow rate after the initiation of step 2. Theflow rate at the end of step 2 is preferably 1.0 to 1.5 times the flowrate at the end of step 1.

In this manner, in step 2, related substance 6 can be detected.

In the present invention, it is possible to add an ion-pair reagent, inaddition to the additives above. Examples of usable ion-pair reagentsinclude sodium alkylsulfonates, such as sodium pentanesulfonate, sodiumhexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, andsodium dodecanesulfonate; sodium alkylsulfates, such as sodium dodecylsulfate; quaternary ammonium salts, such as tetraethyl ammoniumhydroxide, tetrabutyl ammonium hydroxide, tetrabutyl ammonium chloride,and tetrabutyl ammonium bromide; and tertiary amines, such astrihexylamine and trioctylamine.

The pH of the mobile phase (typically the aqueous phase) according tothe present invention can be suitably adjusted with the addition of theadditives mentioned above, and is preferably 2.0 to 5.0.

The detection wavelength usable in the present invention may be 208 to280 nm, preferably 208 to 240 nm, and more preferably 208 to 212 nm, inconsideration of the properties of each related substance.

The temperature of the mobile phase in the column used in the method ofthe present invention may be suitably set. In consideration of theeffect from the external environment, reproducibility, and the like, thetemperature is preferably maintained constant, and is more preferably 25to 50° C., still more preferably 35 to 45° C., and particularlypreferably 40 to 44° C. To maintain the temperature constant, thetemperature of the entire column is controlled, and in addition, apreheated mixer or the like can be used.

In the high-performance liquid chromatography according to the presentinvention, the injection amount, the temperature within the column,etc., may be suitably changed.

Of these related substances, related substances 1 to 5 may besynthesized by known methods or may be obtained from commerciallyavailable products. Related substance 6 may be synthesized fromtrifluridine or commercially available compounds by using known methodsor may be obtained by the method described later. Related substances canbe identified by comparing the retention times in high-performanceliquid chromatography, mass spectra, and results from a photodiode array(PDA) between the thus-obtained related substances and the relatedsubstances detected in accordance with the present invention.

Further, these related substances can be quantitatively measured byeither an external standard method or internal standard method.

When these related substances are possibly contained as impurities in amedicinal drug or pharmaceutical preparation, these related substancesare regulated in accordance with guideline ICH-Q3A of the InternationalCouncil for Harmonisation of Technical Requirements for Pharmaceuticalsfor Human Use. The method of the present invention is very useful sinceit is possible to confirm whether the standard of the guideline issatisfied.

Further, these related substances can be detected from trifluridine or asalt thereof using the method of the present invention. Further yet, inthe present invention, at least one substance selected from relatedsubstances 1 to 6, preferably three or more substances selected fromrelated substances 1 to 6, more preferably four or more substancesselected from related substances 1 to 6, and particularly preferablyrelated substances 2, 3, 5, and 6, can be used as a standard of qualitycontrol.

Trifluridine and trifluridine-related substances used as a standard areof high purity; therefore, the related substances separated under theconditions described above for high-performance liquid chromatographycan be used as a standard. That is, the present invention may also bereferred to as a method for producing the related substances, the methodcomprising separating the related substances from a combination drugcontaining trifluridine or a salt thereof. These related substances maybe related substances 1 to 6 mentioned above, with related substances 2,3, 5, and 6 being preferable.

In the present invention, it is unknown that trifluridine includesrelated substance 2, 3, 5 or 6, and in particular, related substance 6is a novel substance. Therefore, the present invention encompassesrelated substance 2, 3, 5, or 6 used for quality control of apharmaceutical composition containing trifluridine or a salt thereof,and further encompasses related substance 2, 3, 5, or 6 for use as astandard for detecting impurities in a pharmaceutical compositioncontaining trifluridine or a salt thereof. Additionally, the presentinvention also encompasses a method for producing related substance 2,3, 5, or 6, the method comprising separating the related substances anda pharmaceutical composition containing trifluridine or a salt thereoffrom each other.

In the present invention, a method of high-performance liquidchromatography is used to detect trifluridine-related substances.

The method is preferably for detecting at least one trifluridine-relatedsubstance selected from the group consisting of related substances 1 to6 and satisfies the following requirements in steps 1 and 2:

Step 1: the percentage of the organic phase in the entire mobile phaseis 1 to 14% by volume.

Step 2: after step 1, elution is performed by applying a gradient offurther increasing the percentage of the organic phase.

The method more preferably satisfies the following requirements in steps1 and 2:

Step 1: at least one trifluridine-related substance selected from thegroup consisting of related substances 1 to 5 is detected, thepercentage of the organic phase in the entire mobile phase is 1 to 14%by volume, and the operation mode is isocratic.

Step 2: after step 1, elution is performed by applying a gradient offurther increasing the percentage of the organic phase, and relatedsubstance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2:

Step 1: the percentage of the organic phase in the entire mobile phaseis 2 to 10% by volume, the operation mode is isocratic, and relatedsubstances 1 to 5 are detected.

Step 2: after step 1, elution is performed by applying a gradient offurther increasing the percentage of the organic phase, and relatedsubstance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile:

Step 1: the percentage of the organic phase in the entire mobile phaseis 2 to 10% by volume, the operation mode is isocratic, and relatedsubstances 1 to 5 are detected.

Step 2: after step 1, elution is performed by applying a gradient offurther increasing the percentage of the organic phase, the percentageof the organic phase in the entire mobile phase at the end of step 2 is25 to 70% by volume, and related substance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile, the aqueous phase contains water, and the mobile phasecontains phosphate:

Step 1: the percentage of the organic phase in the entire mobile phaseis 2 to 10% by volume, the operation mode is isocratic, and relatedsubstances 1 to 5 are detected.

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase by 0.9% by volume or moreper 1 minute, the percentage of the organic phase in the entire mobilephase at the end of step 2 is 30 to 65% by volume, and related substance6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile, the aqueous phase contains water, and the mobile phasecontains phosphate:

Step 1: the percentage of the organic phase in the entire mobile phaseis 2 to 10% by volume, the operation mode is isocratic, and relatedsubstances 1 to 5 are detected.

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase in the entire mobilephase by 0.9% by volume or more per 1 minute, the percentage of theorganic phase in the entire mobile phase at the end of step 2 is 30 to65% by volume, and related substance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile, the aqueous phase contains water, and the mobile phasecontains phosphate:

Step 1: the percentage of the organic phase in the entire mobile phaseis 3 to 7% by volume, the operation mode is isocratic, and relatedsubstances 1 to 5 are detected.

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase in the entire mobilephase by 0.9% by volume or more per 1 minute, the percentage of theorganic phase in the entire mobile phase at the end of step 2 is 35 to60% by volume, and related substance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile, the aqueous phase contains water, and the mobile phasecontains sodium dihydrogen phosphate.

Step 1: the percentage of the organic phase in the entire mobile phaseis 3 to 7% by volume, the operation mode is isocratic, the measurementtime is 15 to 28 minutes, and related substances 1 to 5 are detected:

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase in the entire mobilephase by 0.9% by volume or more per 1 minute, the percentage of theorganic phase in the entire mobile phase at the end of step 2 is 35 to60% by volume, the measurement time is 10 to 50 minutes, and relatedsubstance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile, the aqueous phase contains water, and the mobile phasecontains sodium dihydrogen phosphate.

Step 1: the percentage of the organic phase in the entire mobile phaseis 3 to 7% by volume, the operation mode is isocratic, the measurementtime is 15 to 28 minutes, and related substances 1 to 5 are detected:

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase in the entire mobilephase by 0.9% by volume or more per 1 minute, the percentage of theorganic phase in the entire mobile phase at the end of step 2 is 35 to60% by volume, the measurement time is 10 to 50 minutes, the flow rateat the end of step 2 is 1.0 to 1.5 times the flow rate in step 1, andrelated substance 6 is detected.

The method more preferably satisfies the following requirements in steps1 and 2, wherein the organic phase in the mobile phase containsacetonitrile, the aqueous phase contains water, and the mobile phasecontains methanol and sodium dihydrogen phosphate:

Step 1: the percentage of the organic phase in the entire mobile phaseis 3 to 7% by volume, the operation mode is isocratic, the measurementtime is 15 to 28 minutes, and related substances 1 to 5 are detected.

Step 2: after step 1, elution is performed by applying a gradient ofincreasing the percentage of the organic phase in the entire mobilephase by 0.9% by volume or more per 1 minute, the percentage of theorganic phase in the entire mobile phase at the end of step 2 is 35 to60% by volume, the measurement time is 10 to 50 minutes, the flow rateat the end of step 2 is 1.0 to 1.5 times the flow rate in step 1, andrelated substance 6 is detected.

EXAMPLES

Measurement was performed by high-performance liquid chromatographyunder the following test conditions.

-   -   Detector: Ultraviolet spectrophotometer (wavelength: 210 nm).    -   Column: An octadecylsilyl silica gel column for liquid        chromatography (3 μm or 5 μm) was placed in a stainless steel        column with an inner diameter of 4.6 mm and a length of 15 cm.    -   Column temperature: 40° C.    -   Flow rate: stated in each Example.    -   Mobile phase: stated in each Example.    -   Gradient: stated in each Example.

The samples to be measured by high-performance liquid chromatographywere prepared as follows.

Trifluridine was dissolved in a solution having the same composition asthat of a mobile phase to be used under each of the measurementconditions, and the resulting product was suitably diluted so that thetrifluridine concentration was about 0.8 mg/mL, thus obtaining samples.

In each Example, the expression “%” in each mobile phase refers to % byvolume.

Example 1-1

Column: Hydrosphere C18, produced by YMC Co., Ltd. (3 μm)

Flow rate: 1.0 mL/min

Mobile phase A: An aqueous solution of 0.05 mol/L sodium dihydrogenphosphate

Mobile phase B: Acetonitrile

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 18 minutes: 95% A/5% B,

18 to 55 minutes: 95% A/5% B→60% A/40% B,

55 to 55.1 minutes: 60% A/40% B→95% A/5% B,

after 55.1 minutes: 95% A/5% B

FIG. 1 shows the measurement results. The results confirmed that theretention time of trifluridine was 17.2 minutes. The results accordingto this measurement method confirmed that trifluridine did not overlapwith valleys or ghost peaks in the baseline.

Example 1-2

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 18 minutes: 95% A/5% B,

18 to 48 minutes: 95% A/5% B→60% A/40% B,

48 to 48.1 minutes: 60% A/40% B→95% A/5% B,

after 48.1 minutes: 95% A/5% B

The conditions of the column, the flow rate, the mobile phase A, and themobile phase B were the same as in Example 1.

The results according to this measurement confirmed that trifluridinedid not overlap with ghost peaks.

Example 1-3

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 20 minutes: 95% A/5% B

20 to 50 minutes: 95% A/5% B→60% A/40% B,

50 to 50.1 minutes: 60% A/40% B→95% A/5% B,

after 50.1 minutes: 95% A/5% B

The conditions of the column, the flow rate, the mobile phase A, and themobile phase B were the same as in Example 1.

FIG. 2 shows the measurement results. The results confirmed that theretention time of trifluridine was 17.1 minutes. The results accordingto this measurement method confirmed that trifluridine did not overlapwith valleys or ghost peaks in the baseline.

Example 1-3-1

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 15 minutes: 95% A/5% B

15 to 45 minutes: 95% A/5% B→60% A/40% B,

45 to 45.1 minutes: 60% A/40% B→95% A/5% B,

after 45.1 minutes: 95% A/5% B

The conditions of the column, the flow rate, the mobile phase A, and themobile phase B were the same as in Example 1.

Column: Inertsil ODS-3, produced by GL Sciences Inc. (3 μm)

The conditions of the flow rate, the mobile phase A, and the mobilephase B were the same as in Example 1-3.

The results according to this measurement confirmed that trifluridinedid not overlap with ghost peaks.

Example 1-3-2

Column: Inertsil ODS-4, produced by GL Sciences Inc. (3 μm)

The conditions of the flow rate, the mobile phase A, the mobile phase B,and the gradient were the same as in Example 1-3-1.

The results according to this measurement confirmed that trifluridinedid not overlap with ghost peaks.

Example 1-3-3

Column: YMC-Pack Pro C18 RS (3 μm)

The conditions of the flow rate, the mobile phase A, the mobile phase B,and the gradient were the same as in Example 1-3-1.

The results according to this measurement confirmed that trifluridinedid not overlap with ghost peaks.

Example 1-3-4

Column: YMC-Pack Pro C18 (3 μm)

The conditions of the flow rate, the mobile phase A, the mobile phase B,and the gradient were the same as in Example 1-3-1.

The results according to this measurement confirmed that trifluridinedid not overlap with ghost peaks.

Example 2-1

Column: Hydrosphere C18, produced by YMC (5 μm)

Flow rate: 0 to 21 minutes: 1.0 mL/min, 21 to 46 minutes: 1.0→1.3mL/min, 46 to 46.1 minutes: 1.3→1.0 mL/min, after 46.1 minute: 1.0mL/min

Mobile phase A: Sodium dihydrogenphosphate dihydrate (3.0 g) wasdissolved in water (1000 mL), and phosphoric acid was added to theresulting mixture to adjust the pH to 2.2, followed by the addition ofmethanol (10 mL).

Mobile phase B: Acetonitrile

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 21 minutes: 96% A/4% B,

21 to 46 minutes: 96% A/4% B→45% A/55% B,

46 to 46.1 minutes: 45% A/55% B→96% A/4% B

after 46.1 minutes: 96% A/4% B

FIG. 3 shows the measurement results. The results confirmed that theretention time of trifluridine was 17.4 minutes. Further, the retentiontime of related substance 1 was confirmed to be 3.1 minutes, theretention time of related substance 2 was confirmed to be 5.6 minutes,the retention time of related substance 3 was confirmed to be 4.1minutes, the retention time of related substance 4 was confirmed to be7.9 minutes, the retention time of related substance 5 was confirmed tobe 8.8 minutes, and the retention time of related substance 6 wasconfirmed to be 42.9 minutes.

The retention time of each of the related substances detected here wasconsistent with the retention time of each related substance separatelypurchased or synthesized, which confirmed that the related substancesdetected here were compounds having the structures of related substances1 to 6.

In conclusion, the results according to this measurement methodconfirmed that trifluridine did not overlap with valleys or ghost peaksin the baseline. The results also confirmed that it was possible toseparate each trifluridine-related substance from trifluridine.Furthermore, the peak of related substance 6 was consistent with theretention time of related substance 6 synthesized below. In view ofthis, the peak at retention time of 42.9 minutes in this measurement wasconsidered to represent related substance 6.

Example 2-2

Column: Unison UK-C18, produced by Imtakt Co p. (5 μm)

The other conditions were the same as in Example 2-1. The conclusion inregard to this measurement was the same as that of Example 2-1.

Example 2-3

Mobile phase A: Sodium dihydrogenphosphate dihydrate (3.0 g) wasdissolved in water (1000 mL), and phosphoric acid was added to theresulting mixture to adjust the pH to 2.2, followed by the addition ofmethanol (9 mL).

The other conditions were the same as in Example 2-1.

The conclusion in regard to this measurement was the same as that ofExample 2-1.

Example 2-4

Flow rate: 0 to 21 minutes: 0.95 mL/min, 21 to 46 minutes: 0.95→1.25mL/min, 46 to 46.1 minutes: 1.25→0.95 mL/min, after 46.1 minutes: 0.95mL/min

The other conditions were the same as in Example 2-1.

The conclusion in regard to this measurement was the same as that ofExample 2-1.

Example 2-5

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 21 minutes: 95.8% A/4.2% B

21 to 46 minutes: 95.8% A/4.2% B→43% A/57% B

46 to 46.1 minutes: 43% A/57% B→95.8% A/4.2% B

after 46.1 minutes: 95.8% A/4.2% B

The other conditions were the same as in Example 2-1.

The conclusion in regard to this measurement was the same as that ofExample 2-1.

Example 3

Column: Inertsil ODS-2, produced by GL Sciences Inc. (5 μm)

Flow rate: 1.0 mL/min

Mobile phase A: An aqueous solution of 0.05 mol/L sodiumdihydrogenphosphate

Mobile phase B: Acetonitrile

Gradient: The mobile phase A and the mobile phase B were controlled byvarying the concentrations as follows:

0 to 2 minutes: 85% A/15% B

2 to 7 minutes: 85% A/15% B→60% A/40% B

after 7 minutes: 60% A/40% B

The measurement results confirmed that the retention time oftrifluridine was 3.6 minutes. Further, the retention times of relatedsubstances 1 to 5 overlapped with each other within a range of 1.0 to2.5 minutes, and the peak areas of these substances could not becalculated. In conclusion, according to this measurement method,although the detection of trifluridine-related substances 1 to 5 was notnecessarily accurate, the detection of related substance 6 was possible.

Example 4

Column: Inertsil ODS-2, produced by GL Sciences Inc. (5 μm)

Flow rate: 1.0 mL/min

Mobile phase A: Water

Mobile phase B: Acetonitrile

Gradient: Isocratic at 60% A/40% B

The measurement results confirmed that the retention time oftrifluridine was 1.8 minutes, and the retention time of relatedsubstance 6 was 7.6 minutes. Although the retention times of relatedsubstances 1 to 5 overlapped with each other within a range of 1.6 to2.5 minutes, and the peak areas of these substances could not becalculated, the detection of related substance 6 was possible.

Reference Example Synthesis of5′-(4-chlorophenylcarboxy)-2′-deoxy-5-trifluoromethyluridine (RelatedSubstance 6)

Commercially available trifluridine (2.00 g) was dissolved in pyridine(8 mL), and p-chlorobenzoylchloride (1.18 g) was slowly added thereto inan ice bath, followed by stirring at room temperature for 1 hour.Thereafter, the solvent was distilled off, and the residue was purifiedby silica gel column chromatography (chloroform/methanol=100/0 to 91/9)to yield 5T-(4-chlorophenylcarboxy)-2′-deoxy-5-trifluoromethyluridine(1.49 g, related substance 6) and3′-(4-chlorophenylcarboxy)-2′-deoxy-5-trifluoromethyluridine (238 mg,reference compound 1).

Related substance 6: 1H-NMR (DMSO-d6, 270 MHz) δ (ppm) 11.91 (1H, s),8.03 (1H, s), 7.95 (2H, d, J=6.75 Hz), 7.60 (2H, d, J=6.75 Hz), 6.07(1H, t, J=6.55 Hz), 5.48 (1H, d, J=4.29 Hz), 4.56-4.38 (3H, m),4.18-4.16 (1H, m)

Reference compound 1: 1H-NMR (DMSO-d6, 270 MHz) δ (ppm) 11.92 (1H, s),8.72 (1H, s), 8.03 (2H, d, J=8.07 Hz), 7.63 (2H, d, J=8.07 Hz), 6.27(1H, t, J=6.75 Hz), 5.48-5.42 (2H, m), 4.28 (1H, s) 3.72 (2H, s).

The invention claimed is:
 1. A method for detection, comprising:subjecting a sample including trifluridine or a salt thereof tohigh-performance liquid chromatography with a mobile phase comprising anorganic phase and an aqueous phase; and detecting related substance 6and at least one of related substances 1 to 5 in the sample by thehigh-performance liquid chromatography, wherein the high-performanceliquid chromatography comprises steps 1 and 2, step 1 is conducted withthe mobile phase including the organic phase in a range of from 1 to 14%by volume, step 2 is conducted after step 1 and includes performingelution by applying a gradient of increasing the percentage of theorganic phase in the mobile phase, the organic phase comprises at leastone solvent and optionally 10% or less water, the aqueous phasecomprises water and optionally 10% or less of at least one solvent, atleast one of the related substances 1 to 5 is detected in the mobilephase of step 1, and the related substance 6 is detected in the mobilephase of step 2, where the related substances 1 to 6 are as follows:related substance 1: 5-carboxyuracil, related substance 2:5-carboxy-2′-deoxy-uridine, related substance 3:2′-deoxy-5-methoxycarbonyluridine, related substance 4:trifluorothymine, related substance 5: 5-methoxycarbonyluracil, andrelated substance 6:5′-(4-chlorophenylcarboxy)-2′-deoxy-5-trifluoromethyluridine.
 2. Themethod according to claim 1, wherein the organic phase is acetonitrile.3. The method according to claim 1, wherein the aqueous phase includesphosphate.
 4. The method according to claim 1, wherein the aqueous phaseincludes methanol.
 5. The method according to claim 1, wherein step 1 isperformed under isocratic conditions.
 6. The method according to claim1, wherein, in step 1, the percentage of the organic phase in the mobilephase is within a range of 2 to 10% by volume.
 7. The method accordingto claim 1, wherein, in step 1, the percentage of the organic phase inthe mobile phase is within a range of 3 to 7% by volume.
 8. The methodaccording to claim 1, wherein the percentage of the organic phase in themobile phase at the end of step 2 is within a range of 25 to 70% byvolume.
 9. The method according to claim 1, wherein the percentage ofthe organic phase in the mobile phase at the end of step 2 is within arange of 30 to 65% by volume.
 10. The method according to claim 1,wherein the percentage of the organic phase in the mobile phase at theend of step 2 is within a range of 35 to 60% by volume.
 11. The methodaccording to claim 1, wherein, in step 2, the elution is performed byapplying a gradient of increasing the percentage of the organic phase inthe mobile phase by 0.9% by volume or more per 1 minute.
 12. The methodaccording to claim 1, wherein, in step 2, the elution is performed byapplying a gradient of increasing the percentage of the organic phase inthe mobile phase by 1.0% by volume or more per 1 minute.
 13. The methodaccording to claim 1, wherein, in step 2, the elution is performed byapplying a gradient of increasing the percentage of the organic phase inthe mobile phase by 2.0% by volume or more per 1 minute.
 14. The methodaccording to claim 1, wherein, in step 2, the elution is performed byapplying a gradient of increasing the percentage of the organic phase inthe mobile phase by 2.0% by volume or more and 5.0% by volume or lessper 1 minute.
 15. The method according to claim 1, wherein step 2 has ameasurement time within a range of 10 to 50 minutes.
 16. The methodaccording to claim 1, wherein a flow rate at the end of step 2 is 1.0 to1.5 times a flow rate in step
 1. 17. A quality control method of acombination drug including trifluridine, comprising: performing themethod of claim 1, wherein the sample is the combination drug.
 18. Amethod of detecting impurities in a combination drug includingtrifluridine, comprising: performing the method of claim 1, wherein thesample is the combination drug.